Expression of plasma platelet-activating factor acetylhydrolase is transcriptionally regulated by mediators of inflammation.
Platelet-activating factor (PAF) is a potent phospholipid with diverse physiological and pathological actions, and it is inactivated by PAF acetylhydrolase. In this study, we analyzed the tissue distribution of the plasma PAF acetylhydrolase mRNA in humans. We isolated a 3.5-kilobase fragment containing the 5' genomic sequence of the plasma PAF acetylhydrolase gene and further characterized the promoter activity. We determined the transcriptional initiation site by primer extension. We then prepared constructs containing various lengths of 5' genomic fragments fused to a luciferase reporter gene and transfected these constructs into COS-7 cells. We found that there is more than one region in the 1.3-kilobase 5' genomic sequence conferring promoter activity and that a very short 5'-flanking region (72 base pairs) is sufficient for more than 65% of the basal activity. In parallel, we examined the regulation of expression of the PAF acetylhydrolase gene. We found that interferon-gamma (IFNgamma) and lipopolysaccharide (LPS) significantly inhibited synthesis of PAF acetylhydrolase, whereas other cytokines, including IFNalpha, interleukin (IL) 1alpha, IL4, IL6, tumor necrosis factor-alpha, granulocyte/macrophage colony-stimulating factor, and macrophage colony-stimulating factor, had a smaller or no effect in human monocyte-derived macrophages. Furthermore, transfection of the promoter/reporter construct into macrophage RAW264.7 cells revealed that IFNgamma and LPS decreased the promoter activity by 35% and 50%, respectively, whereas PAF stimulated it by 52% via its receptor. The promoter activity was much lower in monocytic U937 cells compared with the basal level in COS-7 cells, while the activities in P388D1 and RAW264.7 macrophagic cells were considerably higher than the basal level in COS-7 cells. There are multiple regions in the PAF acetylhydrolase promoter that contain responsive elements for signal transducer and activators of transcription-related proteins, and also for myeloid-specific transcription factors. Our data indicate that the opposite of mRNA expression in monocytes versus macrophages is due to inhibition of the promoter activity in the former and activation in the latter cells.
Cao
Y
Y
Program in Human Molecular Biology and Genetics, the Huntsman Cancer Institute, Salt Lake City, Utah 84112, USA.
Stafforini
D M
DM
Zimmerman
G A
GA
McIntyre
T M
TM
Prescott
S M
SM
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D001483:Base Sequence; D002454:Cell Differentiation / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D016207:Cytokines / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D007371:Interferon-gamma / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000032:analysis / Q000235:genetics*; D010972:Platelet Activating Factor / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D017422:Sequence Analysis, DNA; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics; D014162:Transfection / Q000235:genetics
D016207:Cytokines; D008070:Lipopolysaccharides; D010972:Platelet Activating Factor; D012333:RNA, Messenger; D007371:Interferon-gamma; D010741:Phospholipases A; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D013755:Tetradecanoylphorbol Acetate